Plants of the hemp family, specifically Canabis sativa produce significant amounts of cannabinoids. The most important cannabinoid is .DELTA..sup.9 tetrahydrocannabinol (THC) which produces the psychotrophic effects attributed to marijuana. The exact mechanism of action of THC is still unkown but its effects are primarily on the cardiovasular and central nervous systems.
The most common method for consumption of marijuana is by smoking. .DELTA..sup.9 -THC is rapidly absorbed from the lungs into the blood stream. THC is rapidly metabolized through 11-hydroxy .DELTA..sup.9 THC to a series of polar metabolites with 11-nor-.DELTA..sup.9 THC-carboxylic acid being the primary metabolite. Approximately 80% of a dose of THC is eliminated during the first five days with 80% being excreted in the feces and the remainder in the urine. Depending upon assay sensitivity cannabinoid metabolites may continue to be detected in the urine for up to 10 days in occasional smokers and 36 days in chronic smokers.
In the past, cannabinoids have been detected in biological samples by thin layer chromatography, high pressure liquid chromatography (HPLC), gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), radioimmunoassay or enzyme immunoassay. However, these assay methods are not without drawbacks. Thin layer chromatography is labor intensive and lacks sensitivity. HPLC, GC, and GC/MS are labor intensive, requiring highly trained personnel to carry out extractions of the analyte from the biological matrix, while GC and GC/MS requires a derivatization step as well. Radioimmunoassay reagents degrade spontaneously, require burdensome methods of protecting and monitoring the safety of the personnel involved and generate hazardous waste which must be disposed of in a secure manner. Enzyme immunoassays are subject to variability due to thermal lability of reagents and to matrix effects which alter enzyme activity.
Fluorescence polarization immunoassay procedures provide a reliable quantitative means for measuring the amount of tracer-antibody complex produced in a homogeneous competitive binding assay. Typically, in such a competitive binding immunoassay a ligand (a substance of biological interest to be determined by the technique) competes with a labeled reagent, or "ligand analog," or "tracer" for a limited number of receptor binding sites on antibodies specific to the ligand and ligand analog. The concentration of ligand in the sample determines the amount of ligand analog which binds to the antibody: the amount of ligand analog that will bind is inversely proportional to the concentration of ligand in the sample, because the ligand and the ligand analog each bind to the antibody in proportion to their respective concentrations. Fluorescence polarization techniques are based on the principle that a fluorescent labeled compound, when excited by plane polarized light, will emit fluoresceence having a degree of polarziation inversely related to its rate of rotation. Accordingly, when a tracer-antibody complex having a fluorescent label is excited with plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time that light is absorbed and emitted. In contrast, when an unbound tracer is excited by plane polarized light, its rotation is much faster than that of the corresponding tracer-antibody conjugate. As a result, the light emitted from the unbound tracer molecules is depolarized.
Such fluorescence polarization techniques have been applied in U.S. Pat. No. 4,420,568 to Wang, et al., which is directed to the use of a triazinylamino-fluorescent moiety as the fluorophore.
Cannabinoid antigen conjugates and antibodies have been described in U.S. Pat. No. 4,438,207 to K. Fahrenholt and J. Heveran, in U.S. Pat. No. 4,022,878 to S. Gross, in NIDA Research Monograph No. 7, 28 (1976 ) by Rowley et al., in NIDA Research Monograph No. 7, 15 (1976) by Cook et al., in Nature 249, 154 (1974) by Teale et al., in Nature New Biology 236, 216 (1972) by Grant et al. and in the Journal of Pharmacology-and Pharmaceutics 27, 465, (1975) by Teale et al.
The present invention is an advance in the art in that novel cannabinoid derivative compounds and novel reagents specifically useful in a fluorescence polarization assay are provided. An assay conducted in accordance with the present invention is particularly accurate, as will be explained infra.